Sonic-hedgehog-mediated proliferation requires the localization of PKA to the cilium base.

Cerebellar granular neuronal precursors (CGNPs) proliferate in response to the mitogenic activity of Sonic hedgehog (Shh), and this proliferation is negatively regulated by activation of cAMP-dependent protein kinase (PKA). In the basal state, the PKA catalytic subunits (C-PKA) are inactive because of their association with the regulatory subunits (R-PKA). As the level of cAMP increases, it binds to R-PKA, displacing and thereby activating the C-PKA. Here we report that, in the presence of Shh, inactive C-PKA accumulates at the cilium base of proliferative CGNPs whereas removal of Shh triggers the activation of PKA at this particular location. Furthermore, we demonstrate that the anchoring of the PKA holoenzyme to the cilium base is mediated by the specific binding of the type II PKA regulatory subunit (RII-PKA) to the A-kinase anchoring proteins (AKAPs). Disruption of the interaction between RII-PKA and AKAPs inhibits Shh activity and, therefore, blocks proliferation of CGNP cultures. Collectively, these results demonstrate that the pool of PKA localized to the cilium base of CGNP plays an essential role in the integration of Shh signal transduction.

Gold Tailored Photosensitive Elastin-like Polymer: Synthesis of Temperature, pH and UV-vis Sensitive Probes.

Here, we describe a procedure to manufacture smart hybrid probes that exhibit tunable optical properties as a function of multiple environmental variations. Initially, we achieved a one-pot synthesis of gold-PREP (photo-responsive elastin-like polymer) conjugate Gold-AzoGlu15 via reduction of auric acid in the presence of PREP AzoGlu15. Outstandingly, Gold-AzoGlu15 exhibited pH and temperature sensitiveness. However, Gold-AzoGlu15 was not UV-vis sensitive. We noticed that photo-isomerisation of azobenzene moieties in Gold-AzoGlu15 could not be detected by UV-vis spectroscopy. In a subsequent step, we explored the use of cyclodextrins and the formation of alkanethiol mixed-monolayers over mother Gold-AzoGlu15 by thiol-place exchange reactions in order to decouple photo-isomerisation of azobenzene from the bulk phase absorption. In this sense we achieved the synthesis of ß-cyclodextrin capped Gold-CD-AzoGlu15. Notable was that cis-trans photo-conversion of azobenzene groups in Gold-CD-AzoGlu15 could be successfully detected. Finally, we present the optical properties exhibited by multi-sensitive probe Gold-CD-AzoGlu15 as a function of pH, temperature and UV-vis irradiation. We think that gold-PREP hybrids could be of great interest in the design of multi-functional chromophore-metal nanocomposites that operate in aqueous media for the development of multi-stimuli sensitive detectors for biosensing applications.

Expression of the proneural gene encoding Mash1 suppresses MYCN mitotic activity.

Murine Mash1 (Ascl1) is a member of the basic helix-loop-helix family of transcription factors and has been described to promote differentiation in some neural precursors. The process of differentiation is coordinated with a concomitant cell-cycle arrest, but the molecular mechanism of this process is unclear. Here, we describe for the very first time a direct regulation of an oncogene by a proneural gene. When expressed in proliferating cerebellar granular precursors, expression of the proneural gene encoding Mash1 promotes cell-cycle exit and differentiation, whereas expression of the oncogene MYCN has the opposite effect, promoting the proliferation of these cells in the absence of sonic hedgehog. Moreover, Mash1 overexpression neutralizes MYCN-induced proliferation. We now propose that the mechanism of antagonism between both molecules is based on opposite functions in the transcriptional regulation of the E-box motif, particularly in the E-boxes within the cyclin-D2 promoter, with MYCN acting as a transcriptional activator and Mash1 as a repressor. In agreement with this result, overexpression of cyclin D2 suppressed the anti-proliferative activity of Mash1.

Bone morphogenetic protein 2 opposes Shh-mediated proliferation in cerebellar granule cells through a TIEG-1-based regulation of Nmyc.

Nmyc is a potent regulator of cell cycle in cerebellar granular neuron precursors (CGNPs) and has been proposed to be the main effector of Shh (Sonic hedgehog) proliferative activity. Nmyc ectopic expression is sufficient to promote cell autonomous proliferation and can lead to tumorigenesis. Bone morphogenetic protein 2 (BMP2) antagonizes Shh proliferative effect by promoting cell cycle exit and differentiation in CGNPs. Here we report that BMP2 opposes Shh mitogenic activity by blocking Nmyc expression. We have identified TIEG-1 (KLF10) as the intermediary factor that blocks Nmyc expression through the occupancy of the Sp1 sites present in its promoter. We also demonstrate that TIEG-1 ectopic expression in CGNPs induces cell cycle arrest that can lead to apoptosis but fails to promote differentiation. Moreover, TIEG-1 synergizes with BMP2 activity to terminally differentiate CGNPs and independent differentiator signals such as dibutyryl cAMP and prevents apoptosis in TIEG-1 arrested cells. All together, these data strongly suggest that the BMP2 pathway triggers cell cycle exit and differentiation as two separated but coordinated processes, where TIEG-1 acts as a mediator of the cell cycle arrest.

Bmp2 antagonizes sonic hedgehog-mediated proliferation of cerebellar granule neurones through Smad5 signalling.

During development of the cerebellum, sonic hedgehog (Shh) is directly responsible for the proliferation of granule cell precursors in the external germinal layer. We have looked for signals able to regulate a switch from the Shh-mediated proliferative response to one that directs differentiation of granule neurones. Bone morphogenetic proteins (BMPs) are expressed in distinct neuronal populations within the developing cerebellar cortex. Bmp2 and Bmp4 are expressed in the proliferating precursors and subsequently in differentiated granule neurones of the internal granular layer, whereas Bmp7 is expressed by Purkinje neurones. In primary cultures, Bmp2 and Bmp4, but not Bmp7, are able to prevent Shh-induced proliferation, thereby allowing granule neuron differentiation. Furthermore, Bmp2 treatment downregulates components of the Shh pathway in proliferating granule cell precursors. Smad proteins, the only known BMP receptor substrates capable of transducing the signal, are also differentially expressed in the developing cerebellum: Smad1 in the external germinal layer and Smad5 in newly differentiated granule neurones. Among them, only Smad5 is phosphorylated in vivo and in primary cultures treated with Bmp2, and overexpression of Smad5 is sufficient to induce granule cell differentiation in the presence of Shh. We propose a model in which Bmp2-mediated Smad5 signalling suppresses the proliferative response to Shh by downregulation of the pathway, and allows granule cell precursor to enter their differentiation programme.